Increase In Myelopoiesis Research Articles

Dysregulated myeloid differentiation in colitis is induced by inflammatory osteoclasts in a TNFα-dependent manner

Inflammatory bowel disease (IBD) is characterized by very severe intestinal inflammation associated with extra-intestinal manifestations. One of the most critical ones is bone destruction, which remains a major cause of morbidity and a risk factor for osteopenia and osteoporosis in IBD patients. In various mouse models of IBD, we and other have demonstrated concomitant bone loss due to a significant increase in osteoclast activity. Besides bone resorption, osteoclasts are known to control hematopoietic niches in vivo and modulate inflammatory responses in vitro, suggesting they may participate in chronic inflammation in vivo. Here, using different models of colitis, we showed that osteoclast inhibition significantly reduced disease severity and that induction of osteoclast differentiation by RANKL contributed to disease worsening. Our results demonstrate a direct link between osteoclast activity and myeloid cell accumulation in the intestine during colitis. RNAseq analysis of osteoclasts from colitic mice revealed overexpression of genes involved in the remodeling of hematopoietic stem cell niches. We also demonstrated that osteoclasts induced hematopoietic progenitor proliferation accompanied by a myeloid skewing in the early phases of colitis, which was confirmed in a model of RANKL-induced osteoclastogenesis. Mechanistically, inhibition of TNF-α reduced the induction of myeloid skewing by OCL both in vitro and in vivo. Lastly, we observed that osteoclastic activity and the proportion of myeloid cells in the blood are positively correlated in patients with Crohn’s disease. Collectively, our results shed light on a new role of osteoclasts in colitis in vivo, demonstrating they exert their colitogenic activity through an early action on hematopoiesis, leading to an increase in myelopoiesis sustaining gut inflammation.

Regulation of lymphoid-myeloid lineage bias through regnase-1/3-mediated control of Nfkbiz

AbstractRegulation of lineage biases in hematopoietic stem and progenitor cells (HSPCs) is pivotal for balanced hematopoietic output. However, little is known about the mechanism behind lineage choice in HSPCs. Here, we show that messenger RNA (mRNA) decay factors regnase-1 (Reg1; Zc3h12a) and regnase-3 (Reg3; Zc3h12c) are essential for determining lymphoid fate and restricting myeloid differentiation in HSPCs. Loss of Reg1 and Reg3 resulted in severe impairment of lymphopoiesis and a mild increase in myelopoiesis in the bone marrow. Single-cell RNA sequencing analysis revealed that Reg1 and Reg3 regulate lineage directions in HSPCs via the control of a set of myeloid-related genes. Reg1- and Reg3-mediated control of mRNA encoding Nfkbiz, a transcriptional and epigenetic regulator, was essential for balancing lymphoid/myeloid lineage output in HSPCs in vivo. Furthermore, single-cell assay for transposase-accessible chromatin sequencing analysis revealed that Reg1 and Reg3 control the epigenetic landscape on myeloid-related gene loci in early stage HSPCs via Nfkbiz. Consistently, an antisense oligonucleotide designed to inhibit Reg1- and Reg3-mediated Nfkbiz mRNA degradation primed hematopoietic stem cells toward myeloid lineages by enhancing Nfkbiz expression. Collectively, the collaboration between posttranscriptional control and chromatin remodeling by the Reg1/Reg3-Nfkbiz axis governs HSPC lineage biases, ultimately dictating the fate of lymphoid vs myeloid differentiation.

Reversal of aging-associated increase in myelopoiesis and expression of alarmins by angiotensin-(1–7)

Aging is associated with chronic systemic inflammation largely due to increased myelopoiesis, which in turn increases risk for vascular disease. We have previously shown evidence for the therapeutic potential of Angiotensin-(1–7) (Ang-(1–7)) in reversing vasoreparative dysfunction in aging. This study tested the hypothesis that ischemic vascular repair in aging by Ang-(1–7) involves attenuation of myelopoietic potential in the bone marrow and decreased mobilization of inflammatory cells. Young or Old male mice of age 3–4 and 22–24 months, respectively, received Ang-(1–7) (1 µg/kg/min, s.c.) for four weeks. Myelopoiesis was evaluated in the bone marrow (BM) cells by carrying out the colony forming unit (CFU-GM) assay followed by flow cytometry of monocyte-macrophages. Expression of pro-myelopoietic factors and alarmins in the hematopoietic progenitor-enriched BM cells was evaluated. Hindlimb ischemia (HLI) was induced by femoral ligation, and mobilization of monocytes into the blood stream was determined. Blood flow recovery was monitored by Laser Doppler imaging and infiltration of inflammatory cells was evaluated by immunohistochemistry. BM cells from Old mice generated a higher number of monocytes (Ly6G-CD11b+Ly6Chi) and M1 macrophages (Ly6ChiF4/80+) compared to that of Young, which was reversed by Ang-(1–7). Gene expression of selected myelopoietic factors, alarmins (S100A8, S100A9, S100A14 and HMGb1) and the receptor for alarmins, RAGE, was higher in the Old hematopoietic progenitor-enriched BM cells compared to the Young. Increased expressions of these factors were decreased by Ang-(1–7). Ischemia-induced mobilization of monocytes was higher in Old mice with decreased blood flow recovery and increased infiltration of monocyte-macrophages compared to the Young, all of which were reversed by Ang-(1–7). Enhanced ischemic vascular repair by Ang-(1–7) in aging is largely by decreasing the generation and recruitment of inflammatory monocyte-macrophages to the areas of ischemic injury. This is associated with decreased alarmin signaling in the BM-hematopoietic progenitor cells.

Toll-like receptor 9 deficiency induces osteoclastic bone loss via gut microbiota-associated systemic chronic inflammation

Toll-like receptors (TLRs) play pivotal roles in inflammation and provide important links between the immune and skeletal systems. Although the activation of TLRs may affect osteoclast differentiation and bone metabolism, whether and how TLRs are required for normal bone remodeling remains to be fully explored. In the current study, we show for the first time that TLR9−/− mice exhibit a low bone mass and low-grade systemic chronic inflammation, which is characterized by the expansion of CD4+ T cells and increased levels of inflammatory cytokines, including TNFα, RANKL, and IL1β. The increased levels of these cytokines significantly promote osteoclastogenesis and induce bone loss. Importantly, TLR9 deletion alters the gut microbiota, and this dysbiosis is the basis of the systemic inflammation and bone loss observed in TLR9−/− mice. Furthermore, through single-cell RNA sequencing, we identified myeloid-biased hematopoiesis in the bone marrow of TLR9−/− mice and determined that the increase in myelopoiesis, likely caused by the adaptation of hematopoietic stem cells to systemic inflammation, also contributes to inflammation-induced osteoclastogenesis and subsequent bone loss in TLR9−/− mice. Thus, our study provides novel evidence that TLR9 signaling connects the gut microbiota, immune system, and bone and is critical in maintaining the homeostasis of inflammation, hematopoiesis, and bone metabolism under normal conditions.

Cdc42 in osterix-expressing cells alters osteoblast behavior and myeloid lineage commitment.

Osteoblasts are not only responsible for bone formation. They also support hematopoiesis. This requires responding to cues originating from several signaling pathways, a task performed by Rho GTPases. We therefore examined several transgenic mouse models and used inhibitors of Cdc42 in vitro. Deletion of Cdc42 in vivo using the Osterix promoter suppressed osteoblast function, while its deletion in differentiating osteoblasts using the Collagen-α1(I) promoter decreased osteoblast numbers. In both cases, bone mineral density diminished confirming the importance of Cdc42. Evaluation of hematopoiesis revealed that deletion of Cdc42 using the Osterix, but not the Collagen-α1(I) promoter increased the common myeloid progenitors (CMPs) in the bone marrow as well as the erythrocytes and the thrombocytes/platelets in peripheral blood. Causality between Cdc42 loss in early osteoblasts and increased myelopoiesis was confirmed in vitro. Work in vitro supported the conclusion that interleukin-4 mediated the increase in myelopoiesis. Thus, Cdc42 is required for healthy bone through regulation of bone formation in Osterix-expressing osteoblasts and the number of osteoblasts in differentiating osteoblasts. In addition, its expression in early osteoblasts/stromal cells modulates myelopoiesis. This highlights the importance of osteoblasts in regulating hematopoiesis.

Immunomodulatory activity in tumor-bearing mice treated with Withania somnifera extract

We investigated some actions of Withania somnifera on the growth and differentiation of hematopoietic precursors [granulocyte/macrophage colony cell formation (CFU-GM)] of normal animals and EAT bearers, which were treated with different doses (20, 50, or 100 mg/kg/day). We also evaluated the presence of colony stimulatory factors in the animal's serum, as well as its survival. Furthermore, we analyzed lymphocyte proliferation, IFN-ɤ, and TNF-α concentrations in treated bearing mice. Our results demonstrated Withania somnifera effectiveness on hematopoietic precursors growth and differentiation in marrow and spleen TAE-bearing mice. As it was already expected, EAT produced myelosuppression and increased CFU-GM spleen number concomitantly. The treatment of EAT-bearing animals with W.S. (20, 50, and 100 mg/Kg) produced a dose-dependent increase in myelopoiesis, an increase in a lifetime, and a reduction in spleen colony number. All this happened parallel to survival. As to lymphocyte proliferation, they were also dose-dependent in treated bearing animals. Concerning IFN-γ levels, we observed a significant reduction in non-treated bearing mice. Levels of TNF-α of treated bearing mice significantly increased when compared to the non-treated bearing group. These results are encouraging since they favor the use of W.S. extract in therapeutic combinations with other chemotherapeutic agents to reduce myelotoxicity and supplement the tumoricidal efficacy of this plant.

GM-CSF drives dysregulated hematopoietic stem cell activity and pathogenic extramedullary myelopoiesis in experimental spondyloarthritis

Dysregulated hematopoiesis occurs in several chronic inflammatory diseases, but it remains unclear how hematopoietic stem cells (HSCs) in the bone marrow (BM) sense peripheral inflammation and contribute to tissue damage in arthritis. Here, we show the HSC gene expression program is biased toward myelopoiesis and differentiation skewed toward granulocyte-monocyte progenitors (GMP) during joint and intestinal inflammation in experimental spondyloarthritis (SpA). GM-CSF-receptor is increased on HSCs and multipotent progenitors, favoring a striking increase in myelopoiesis at the earliest hematopoietic stages. GMP accumulate in the BM in SpA and, unexpectedly, at extramedullary sites: in the inflamed joints and spleen. Furthermore, we show that GM-CSF promotes extramedullary myelopoiesis, tissue-toxic neutrophil accumulation in target organs, and GM-CSF prophylactic or therapeutic blockade substantially decreases SpA severity. Surprisingly, besides CD4+ T cells and innate lymphoid cells, mast cells are a source of GM-CSF in this model, and its pathogenic production is promoted by the alarmin IL-33.

IMPACT-TB*: A Phase II Trial Assessing the Capacity of Low Dose Imatinib to Induce Myelopoiesis and Enhance Host Anti-Microbial Immunity Against Tuberculosis. *Imatinib Mesylate per Oral As a Clinical Therapeutic for TB

Background: With existing anti-tubercular therapies, treatment success rates for multi- and extensively-drug resistant tuberculosis (MDR-TB, XDR-TB) are dismal, highlighting the urgent need for new drugs. Ratios of myeloid to lymphoid cells are important predictors of TB disease susceptibility and vaccine efficacy. Imatinib mesylate (Gleevec), a cancer drug used in humans for chronic myelogenous leukemia (CML) and gastrointestinal stromal tumors (GISTs), is a potential "host- directed therapeutic (HDT)" for drug resistant TB infections and HIV/TB co-infections. Imatinib inhibits c-Abl tyrosine kinase (TK), which is dysregulated in CML, as well as related TKs (e.g. c-Kit). Imatinib facilitates clearance of Mycobacterium tuberculosis (Mtb), by disrupting the cellular mechanisms that Mtb uses for entry and survival in host cells. At doses 10-fold lower than those used for CML, imatinib also stimulates "emergency hematopoiesis," a host immune response to infection that mobilizes myeloid cell differentiation in the bone marrow and alters ratios of myeloid to lymphoid cells in the periphery towards protective levels, an effect suppressed by Mtb. Imatinib acts synergistically with antibiotics and is effective against antibiotic-resistant mycobacteria, and may be less likely to engender resistance compared to antibiotics. Preclinical data at low doses indicate efficacy of imatinib in a non-human primate (NHP) model of infection with TB or, alternatively, TB/SIV, which mimics poorly controlled TB or HIV/TB in humans. Study Design, Endpoints, and statistical design: We describe the design of two NIH-sponsored Phase II clinical trials in humans (NCT03891901). The first trial will comprise testing of 4 low doses of imatinib with or without antibiotics in 72 normal individuals in the United States. We will determine safety, immunological responses, and drug-drug interactions with standard of care antibiotics. The primary endpoints are to (i) identify the two best doses that induce myelopoiesis in humans, (ii) maximize bactericidal activity by immune cells ex vivo, and (iii) maximize safety. The trial is powered to detect a 2-fold increase in myelopoiesis across all imatinib doses relative to untreated individuals. The second trial will assess the safety and microbiologic efficacy of two doses of imatinib administered over 3 months in conjunction with optimized background antibiotic regimens versus the optimized background regimen alone. The trial will include 180 individuals with TB or HIV/TB in South Africa (60 per arm). The primary endpoints are (i) the time to sputum culture conversion, (ii) lung function by PET-CT and spirometry, and (iii) immunological parameters associated with myelopoieisis. Secondary endpoints include transcriptomics, proteomics, and measures of pathogen-specific immune responses. The trial is powered to detect a 2-fold increase in myelopoiesis across all imatinib doses relative to untreated individuals. The trial is statistically powered to distinguish at either dose an improvement over baseline treatment in time to sputum conversion or lung function or both. Major Inclusion/Exclusion Criteria: Phase II dosing trial: Normal volunteers, age 18 - 55 years. Phase II efficacy trial: Active antibiotic-sensitive tuberculosis with or without HIV. Pregnant women are excluded from both studies. Trial Status : The dosing trial has FDA and NIAID approval to begin and is currently undergoing registration with DAIDS prior to starting enrollment. Overall Goal: Data from these trials will provide a paradigm with which to further evaluate imatinib or other immunomodulatory HDTs as therapeutics for TB infection and HIV/TB co-infection. If successful, imatinib may shorten the time of treatment and limit development of antibiotic resistance, and may be useful against antibiotic resistant strains. This project is supported a grant from NIH/NIAID. Disclosures Waller: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Other: Travel expenses, Research Funding; Cerus Corporation: Other: Stock, Patents & Royalties; Chimerix: Other: Stock; Cambium Oncology: Patents & Royalties: Patents, royalties or other intellectual property ; Amgen: Consultancy; Kalytera: Consultancy. OffLabel Disclosure: Imatinib is known as a therapeutic for CML. Here, we will discuss our plans to develop imatinib as a host-directed-therapy to improve treatment of multi-drug-resistant tuberculosis

Plasma Cells Are Obligate Effectors of Enhanced Myelopoiesis in Aging Bone Marrow

Aging results in increased myelopoiesis, which is linked to the increased incidence of myeloid leukemias and production of myeloid-derived suppressor cells. Here, we examined the contribution of plasma cells (PCs) to age-related increases in myelopoiesis, as PCs exhibit immune regulatory function and sequester in bone marrow (BM). PC number was increased in old BM, and they exhibited high expression of genes encoding inflammatory cytokines and pathogen sensors. Antibody-mediated depletion of PCs from old mice reduced the number of myeloid-biased hematopoietic stem cells and mature myeloid cells to levels in young animals, but lymphopoiesis was not rejuvenated, indicating that redundant mechanisms inhibit that process. PCs also regulated the production of inflammatory factors from BM stromal cells, and disruption of the PC-stromal cell circuitry with inhibitors of the cytokines IL-1 and TNF-α attenuated myelopoiesis in old mice. Thus, the age-related increase in myelopoiesis is driven by an inflammatory network orchestrated by PCs.

T-cell-Secreted TNFα Induces Emergency Myelopoiesis and Myeloid-Derived Suppressor Cell Differentiation in Cancer.

Hematopoiesis in patients with cancer is characterized by reduced production of red blood cells and an increase in myelopoiesis, which contributes to the immunosuppressive environment in cancer. Some tumors produce growth factors that directly stimulate myelopoiesis such as G-CSF or GM-CSF. However, for a majority of tumors that do not directly secrete hematopoietic growth factors, the mechanisms involved in the activation of myelopoiesis are poorly characterized. In this study, we document in different murine tumor models activated hematopoiesis with increased proliferation of long-term and short-term hematopoietic stem cells and myeloid progenitor cells. As a consequence, the frequency of myeloid-derived suppressor cells and its ratio to CD8+ T cells increased in tumor-bearing mice. Activation of hematopoiesis and myeloid differentiation in tumor-bearing mice was induced by TNFα, which was mainly secreted by activated CD4+ T cells. Therefore, the activated adaptive immune system in cancer induces emergency myelopoiesis and immunosuppression. SIGNIFICANCE: These findings characterize a regulatory circuit linking activated T cells to suppression of tumor-specific immune responses, providing a conceptual advance in the understanding of emergency-hematopoiesis in cancer and opening new targets for therapeutic approaches. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/2/346/F1.large.jpg.

Long-lived plasma cells regulate patterns of hematopoiesis in aging.

Abstract In both humans and mice, aging results in significant alterations to the patterns of bone marrow hematopoiesis. This is highlighted by a precipitous reduction in B lymphopoiesis while myelopoiesis is enhanced. Treatment of young mice with factors such as GM-CSF or IL-1 can skew hematopoiesis towards myeloid development. This suggests that imbalances in the relative concentrations of various cytokines such as inflammatory mediators, whose production is thought to increase with age, could contribute to suppressed lymphoid and enhanced myeloid development in aging. In this case, the resulting stimulation of myelopoiesis would dominate the limited number of niches in the bone marrow environment at the expense of lymphopoiesis. However, whether the age-related decline in lymphopoiesis and increase in myelopoiesis are linked or represent distinct processes is not known. We observed that long-lived plasma cells from old mice acquire an inflammatory gene signature and accumulate in the bone marrow. Addition of long-lived plasma cells to long-term in vitro cultures demonstrated the ability of these cells to promote granulopoiesis from hematopoietic stem cells as well as myeloid progenitors. However, while depletion of long-lived plasma cells in old mice by injection of anti-CD138 antibodies resulted in a significant reduction in myeloid development, B lymphopoiesis remained suppressed. These data demonstrate a previously unappreciated ability of inflammatory plasma cells to stimulate myelopoiesis and provide evidence that, in the context of aging, enhanced myelopoiesis and depressed lymphopoiesis are distinct, independently regulated processes.

Modulation of Myelopoiesis Progenitors Is an Integral Component of Trained Immunity

SummaryTrained innate immunity fosters a sustained favorable response of myeloid cells to a secondary challenge, despite their short lifespan in circulation. We thus hypothesized that trained immunity acts via modulation of hematopoietic stem and progenitor cells (HSPCs). Administration of β-glucan (prototypical trained-immunity-inducing agonist) to mice induced expansion of progenitors of the myeloid lineage, which was associated with elevated signaling by innate immune mediators, such as IL-1β and granulocyte-macrophage colony-stimulating factor (GM-CSF), and with adaptations in glucose metabolism and cholesterol biosynthesis. The trained-immunity-related increase in myelopoiesis resulted in a beneficial response to secondary LPS challenge and protection from chemotherapy-induced myelosuppression in mice. Therefore, modulation of myeloid progenitors in the bone marrow is an integral component of trained immunity, which to date, was considered to involve functional changes of mature myeloid cells in the periphery.

Hematopoietic Stem Cell Transcription Factor PU.1 with Mutated β3/β4 Domain Selectively Elicits Myeloid Differentiation

AbstractIn many hematopoietic malignancies such as lymphomas & leukemias, aberrant differentiation is the major feature of malignant phenotype that often results from a single genetic alteration and provides a site-specific target for therapy. Therefore, targeting of protein-protein interactions that have been identified as mediators of transcriptional repression that blocks normal hematopoietic differentiation holds great promise for therapeutic applications. An example is GATA-1, a critical erythroid transcription factor that is capable of suppressing the myeloid phenotype by down-regulating PU.1 in a dose-dependent manner. PU.1 is the most crucial transcription factor known to be required for myeloid differentiation of hematopoietic stem cells or progenitor cells. Its reduced expression correlates with a bad prognosis and immature phenotype in acute myeloid leukemia (AML). GATA-1 downregulates PU.1 by interacting though its C-terminal zinc finger region with the β3/β4 region of PU.1 and displacing its coactivator c-Jun. We hypothesize that disruption of PU.1-GATA-1 interaction by mutating the β3/β4 region of PU.1 may prevent its GATA-1-mediated repression, which in turn will upregulate PU.1 expression and hence myelopoiesis. Our analysis of the PU.1 mutants, Lys240Arg and Tyr244Ala, revealed that they exhibit an increase in myelopoiesis in vitro. Thus our data have implications for the prospect of targeting PU.1-GATA-1 interaction for therapeutic intervention.

MIR-23A microRNA cluster inhibits B-cell development

The transcription factor PU.1 (encoded by Sfpi1) promotes myeloid differentiation, but it is unclear what downstream genes are involved. Micro RNAs (miRNAs) are a class of small RNAs that regulate many cellular pathways, including proliferation, survival, and differentiation. The objective of this study was to identify miRNAs downstream of PU.1 that regulate hematopoietic development. miRNAs that change expression in a PU.1-inducible cell line were identified with microarrays. The promoter for an miRNA cluster upregulated by PU.1 induction was analyzed for PU.1 binding by electrophoretic mobility shift and chromatin immunoprecipitation assays. Retroviral transduction of hematopoietic progenitors was performed to evaluate the effect of miRNA expression on hematopoietic development in vitro and in vivo. We identified an miRNA cluster whose pri-transcript is regulated by PU.1. The pri-miRNA encodes three mature miRNAs: miR-23a, miR-27a, and miR-24-2. Each miRNA is more abundant in myeloid cells compared to lymphoid cells. When hematopoietic progenitors expressing the 23a cluster miRNAs were cultured in B-cell-promoting conditions, we observed a dramatic decrease in B lymphopoiesis and an increase in myelopoiesis compared to control cultures. In vivo, hematopoietic progenitors expressing the miR-23a cluster generate reduced numbers of B cells compared to control cells. The miR-23a cluster is a downstream target of PU.1 involved in antagonizing lymphoid cell fate acquisition. Although miRNAs have been identified downstream of PU.1 in mediating development of monocytes and granulocytes, the 23a cluster is the first downstream miRNA target implicated in regulating development of myeloid vs lymphoid cells.

Tel/PDGFRβ inhibits self-renewal and directs myelomonocytic differentiation of ES cells

The leukemic oncogene Tel/PDGFRβ, was inducibly expressed in embryonic stem (ES) cells and the phenotypic and molecular changes occurring during hematopoietic differentiation investigated. Expression of Tel/PDGFRβ resulted in an inability of ES cells to self-renew and caused a significant increase in myelopoiesis with a corresponding decrease in erythropoiesis. Analysis of gene expression patterns indicated a dramatic alteration in the levels of genes associated with self-renewal and differentiation, especially myelomonocytic genes in Tel/PDGFRβ-expressing cells. This study indicates Tel/PDGFRβ drives myelopoiesis by altering expression of genes involved in hematopoiesis and demonstrates the potential of this stem cell system to study oncogene-induced pathogenesis.

Antitumour activity of [1,2-di(cyclopentadienyl)-1,2-di( p- N, N-dimethylaminophenyl)-ethanediyl] titanium dichloride in xenografted Ehrlich's ascites tumour

The effects of a new titanocene compound with an ansa ligand in the cyclopentadienyl rings, the 1,2-di(cyclopentadienyl)-1,2-di( p- N, N-dimethylaminophenyl)-ethanediyl] titanium dichloride (TITANOCENE X), on the growth and differentiation of granulocyte–macrophage progenitor cells [colony-forming unit-granulocyte–macrophage (CFU-GM)] and Natural killer (NK) cell activity in Ehrlich's ascites tumour (EAT)-bearing mice were studied. Myelosuppression concomitant with increased numbers of spleen CFU-GM was observed in tumour-bearing mice. Treatment of these animals with TITANOCENE X (2.5–50 mg/kg/day) produced an increase in myelopoiesis, in a dose-dependent manner, and reduced spleen colony formation. In addition, the treatment of EAT-bearing mice with 3 doses of 20 or 50 mg/kg TITANOCENE X restored to normal values the reduced Natural killer cell function observed during tumour growth. In parallel, TITANOCENE X prolonged, in a dose-dependent manner, the survival of mice inoculated with Ehrlich's ascites tumour. The highest dose of 50 mg/kg prolonged in 50% the survival time of EAT-bearing mice, compared to non-treated tumour-bearing controls. In comparison with previous results from our laboratory addressing the effects of titanocenes on haematopoiesis, we observed with TITANOCENE X a similar effective profile as for bis(cyclopentadienyl) dithiocyanate titanium(IV), being both less effective than di(cyclopentadienyl) dichloro titanium(IV), since the latter not only prolonged, but also increased the rate of survival. These differences in efficacy may be due to the nature of the ansa-cyclopentadienyl ligand used in TITANOCENE X, since the C 2 bridge between the two cyclopentadienyl groups will increase the hydrolytic stability by an organometallic chelate effect. Also, the introduction of two dimethylamino substituents increases the water solubility of TITANOCENE X when compared to titanocene dichloride itself.

The Role of DNA Methylation of HoxA5, a Regulator of Haematopoiesis, in Progression to Myeloid Blast Crisis in CML.

Hox (homeobox) genes are known to be key regulators of development and haematopoiesis and several have also been implicated in leukaemogenesis. Overexpression of HoxA5 in human haematopoietic progenitors leads to an increase in myelopoiesis, suggesting a role for this hox gene during induction of myeloid differentiation. Inactivation of genes by CpG island DNA methylation is known to be important in the development and progression of leukaemia, and inhibitors of DNA methylation are currently of great interest as novel therapeutics for a number of haematopoietic malignancies. Here we show that in peripheral blood from healthy volunteers HoxA5 exhibits methylation of 50% of alleles across an extensive CpG island covering the promoter region/1st exon of HoxA5. In patients with chronic myeloid leukaemia, 33% (15/45 patients) exhibited increased methylation of HoxA5 (80–100% of alleles methylated) in the chronic phase of the disease. However, such hypermethylation of HoxA5 was invariably present in samples from patients in myeloid blast crisis (15/15 patients). In contrast, patients in lymphoid blast crisis did not exhibit increased levels of hypermethylation. Analysis of patients in chronic phase demonstrated a statistically significant correlation (p < 0.006) between hypermethylation of the HoxA5 gene and other prognostic factors associated with high risk of progression to blast crisis (high Hasford/Sokal score, incomplete response to Imatinib, known early progression, del 9), suggesting that methylation of HoxA5 may be a clinically useful prognostic indicator. The results are also compatible with a direct role for hypermethylation of HoxA5, and consequent loss of HoxA5 expression, in inhibition of myeloid differentiation during progression to blast crisis and implicate HoxA5 as a therapeutic target for inhibitors of DNA methylation in the treatment of leukaemia.

The effect of a Titanocene Dichloride derivative, Ti IV (C 5H 5) 2 NCS 2, on the haematopoietic response of Ehrlich tumour-bearing mice

The effects of the [Ti IV (C 5H 5) 2 NCS 2] metallocene (BCDT), a Titanocene Dichloride derivative, on the growth and differentiation of granulocyte-macrophage progenitor cells [colony-forming unit-granulocyte-macrophage (CFU-GM)] and bone marrow cellularity in normal and Ehrlich ascites tumour-bearing mice were studied. As expected for the Ehrlich ascites tumour-model, concomitant myelosuppression, increased number of spleen CFU-GM and changes in bone marrow cellularity were observed. The treatment of Ehrlich ascites tumour-bearing mice with BCDT (10–30 mg/kg/day) produced a dose-dependent increase in myelopoiesis, a reduction in splenic colonies and a restoration in the total and differential marrow cell counts. We also observed an increase in CFU-GM number when bone marrow cells obtained from normal mice were incubated in vitro with serum from normal mice treated with BCDT. In addition, BCDT prolonged, in a dose-dependent manner, the survival of mice inoculated with Ehrlich ascites tumour. Although it has been previously reported that substitutions in the two halides of the titanocene do not interfere with antitumoural effect, our results with BCDT demonstrated a reduction in antitumour efficacy when compared to previous results with the original titanocene produced in our laboratory.

Haemopoietic cell responses in the blood and bone marrow of sheep infected with the abomasal nematode Telodorsagia circumcincta

The generation of bone marrow and blood haemopoietic progenitor colony-forming cells (CFCs) in sheep given primary or challenge infections with the nematode parasite Telodorsagia circumcincta is described. Ten days after a primary infection, the frequency of early multipotential-CFCs, eosinophil-CFCs, macrophage-CFCs and mast cell or basophil-CFCs was greater than in controls. These frequencies then declined to pre-infection levels by day 21. Blood CFCs (mainly macrophage-CFCs and eosinophil-CFCs) also increased after infection, indicating a migration of CFCs, presumably to the site of infection. Ten days after challenge infection there was less marked myelopoiesis than in the primary infection on day 10, though both eosinophil-CFCs and mast cell or basophil-CFCs were significantly above control values. Blood CFC output (mainly macrophage-CFCs and eosinophil-CFCs) reached a peak 2-6 days after challenge, evidence of rapid recruitment to the site of infection. Telodorsagia circumcincta infection is therefore associated with an increase in myelopoiesis, particularly for the cell types characteristic of the local inflammatory response to abomasal nematodes. There was no correlation between any of the haemopoietic cell responses measured and worm burdens in individual animals after either primary or challenge infection.

Role of colony-stimulating factor in myelopoiesis in murine long-term bone marrow cultures

Weekly medium change or midweek feeding of long-term bone marrow cultures (LTMBCs) results in a significant increase in total myeloid cell production. Proliferative myeloid cells peak 48 hours after feeding, and nonproliferative myeloid cells reach maximum levels at 72 hours. This increase in myelopoiesis is invariably preceded by a significant elevation in biologically and immunologically measurable colony-stimulating factor (CSF) in the supernatants of LTBMC. The level peaks 24 hours after medium change, then gradually returns to basal values. The decrease in CSF relates to its consumption by generating myeloid precursors because no fluctuation in the levels occur in cultures without active myelopoiesis. No significant inhibitors or promoters of CSF were detected. When highly purified L cell CSF, CSF in lung-conditioned medium, or CSF concentrated from LTBMC supernatant is added to cultures, an identical increase in myelopoiesis occurs. Anti- CSF antiserum, added to culture at the time of medium change, totally neutralizes supernatant CSF levels but does not affect myelopoiesis. These findings suggest a potential regulatory role for CSF in myelopoiesis in LTBMC. CSF appears to function within the microenvironment through a mechanism involving cell:cell interactions or by causing the production of other substances that stimulate myelopoiesis. Because exogenous CSF stimulates myelopoiesis, it is likely that it too can react either directly or through microenvironmental cells to stimulate primitive myeloid cells to divide.